| Inner Mongolia Cashmere goat has been as well known as its cashmere quality with quality of the velvet soft texture, light. While the development of meat and meat prouduct,most focus will be put on the quality of cashmere meat. At the same time, it will produce important influence and the certain economic efficiency. Lamb as an important part of meat consumption, its nutritional value is paid more and more attention, different muscles also exist certain differences, like both physical and chemical indicators, protein composition. Deeply study of the differences in cashmere goat skeletal muscle are guiding the development of animal husbandry. This research use the routine nutritional ingredient analysis method in three parts of the skeletal muscles of cashmere goats and the physico-chemical properties of the object of study, to construct transcriptome library, using Illumina HiSeq 2000 sequencing high-throughput sequencing platforms; sequencing and goats reference genome alignment after fully reveal characteristic transcriptome library, including gene expression, gene annotation, genetic differences, alternative splicing, new transcripts, cSNP etc; establish two-dimensional electrophoresis and mass spectrometry platform to carry out research, differences in muscle tissue of cashmere goat, and made a further study on the part of the difference in the level of gene expression of protein, protein gene and the difference on the part of the difference obtained by RT-PCR method for the relative quantitative analysis, to verify the reliability of the data from high-throughput sequencing. Results are as follows:1,The physicochemical analysis on three parts of skeletal muscle in cashmere goat showed that the highest content of protein was in longissimus muscle, the highest content of ash was in the triceps muscle of arm, the highest content of fat and moisture was in luteal muscle;The results of determination about amino acid content on three muscles, the highest total amino acid content was in the longissimus muscle, the sequence of the first four parts of the highest total amino acids as: aspartic acid glutamic acid,leucine lysine(the average content of more than 7%), the highest proportion of umami amino acids was the triceps muscle of arm; The determination results of fatty acid on the three parts showed that the content of IMF and monounsaturated fat acid content was the highest in the gluteus; The lowest content of polyunsaturated fatty acid was in the longissimus muscle.2, The establishment of mRNA level analysis platform by transcripting for three skeletal muscle in cashmere goat rechieved 18.63 GB of data, 92278723 reads of double end;The rate of matching efficiency with the reference gene was above 80%; mining to 631 new genes could be annotated to function, there are 433 genes, among them 415 genes annotated with NR, 389 genes annotated with SwissProt, 274 genes could be annotated with GO, 71 genes could be annotated with KEGG, 15 genes could be annotated with COG classification of functional annotation of the corresponding gene expression; sample quantity and analysis of differentially expressed genes, and the differentially expressed genes the model of clustering, functional annotation and enrichment analysis.3, According to FDR≤0.001 and│log2Ratio│≥1,one compared with another as cross reference of the longissimus dorsi muscle in the three parts of the transcriptome database.The results showed that there were 790 differentially expressed genes between longissimus dorsi and triceps muscle of arm, the latter relative to the former 342 gene expression in the triceps muscle of arm under the regulation, the up-regulated expression 448 gene; As FDR FDR ≤0.001and│log2Ratio│≥5were selected, we got 10 gene expression down regulated significantly, including 4 new genes for goat, upregulation of 21 genes, including 1 new gene for goats; according to the longissimus dorsi muscle and hip muscle between 571 differentially expressed genes, the latter relative to the former 214 downregulated genes, up-regulated 357 genes; triceps muscle of arm and gluteal muscle between the 300 differentially expressed genes, the latter relative to the former 139 downregulated genes, up-regulated 161 genes.4, The classification of differentially expressed genes annotated by three kinds of skeletal muscle two two alignment, the result was found that there were 735 notes to the function, of which there are 716 genes could be annotated with SwissProt, 639 genes could be annotated with GO, 393 genes could be annotated with KEGG, 242 genes could be annotated with COG classification. The gene specific differences obtained for COG classification and annotation to filter the fleshy related gene discovery, triceps muscle of arm relative back differences longest muscle genes in 114 genes associated with meat, 81 up-regulated genes, 33 genes down regulated. The difference of gene is mostly associated with skeletal muscle development related, followed by muscle development, these comments basically includes the actin cytoskeleton, cycle regulation, cell differentiation and synthesis, signal transduction and fat transport and metabolism related functions. The up-regulated genes, MYH2 and MLC3 belong to the actin(Actomyosin), in addition there are 67 genes belong to myosin light chains(Myosin light chain), abbreviated as MLC. In the down regulated genes in the 8 genes belong to MLC, 27 belong to the skeletal muscle, 1 belong to the muscle.5, The experimential conditions of two-dimensional gel electrophoresis in skeletal muscle tissue of cashmere goat were analyzed and optimized by different experimental conditions, including different sample extracting and purifing, different pH IPG strips,different loading quantities, different isoelectric focusing process et al. Meanwhile, the common problems occurring in 2-DE experience were analyzed. The results showed that the process of grinding in liquid nitrogen and following by ultrasonication, the effect of sample purification with 2-DE clean up kit, pH 4-7 17 cm pH IPG strip, the 300 ug loading quantity, isoelectric focusing process repeated several times in addition to salt step, maps were of good quality. The results indicated that the 2-DE maps of cashmere goat muscle were obtained with high resolution and reproducibility, and can be used for the further proteome analysis.Using mass spectrometry analysis obtained the differences of 21 cashmere goats and three sites between skeletal muscle protein, in which 17 proteins were associated with goat muscle development.It was not founded to be associated with glycolysis related protein in three kinds of skeletal muscle. Muscle differentiation was explained by oxidative metabolism related protein regulation. In addition, the further study about differences among the proteins showed the red muscle fiber content in three parts of the skeletal muscle was higher.Triceps muscle of arm in the expression level of MLC2 was higher than that of the other two parts, explaining the white muscle fiber relatively high more than the others. Collagen content in triceps muscle of arm was higher than the longissimus dorsi and gluteal muscle, which means the growth of triceps muscle of arm was so faster. The content of the red muscle fiber ingluteal muscles and longissimus dorsi muscle was higher than that of brachial triceps, it meaned their tenderness was better.6, This study ultilized the analysis of the Real-time gene level on the difference of the resulting protein, which founded that the identification of gene in MLC2 gene expression in four parts of the highest, followed by MYLPF, MB, SOD1, GOT2. The highest volume and significantly the expression of MLC2 in the triceps muscle of arm in(P<0.001), followed by the stock two myo, longissimus dorsi muscle and the gluteus; MYLPF gene expression was the highest in the gluteal muscle, followed by the longissimus muscle, expression in the triceps muscle of arm and the stock two myo in relatively low; MB gene the highest expression level in the triceps muscle of arm and the difference was significant, followed by unit two biceps, longissimus dorsi muscle and the gluteus; distribution of the expression level of SOD gene is the arm triceps, gluteal muscle, the stock two myo and longissimus muscle; GOT is the arm triceps, longissimus muscle, gluteus and stock two myo. |
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